PROJECT SUMMARY Approximately 20% of non-small cell lung cancer (NSCLC) patients with EGFR mutations harbor atypical mutations. Unlike NSCLC patients bearing ?classical? EGFR mutations (L858R or exon 19 deletions), the patients with atypical mutations in exon 18 or 20 are resistant to FDA-approved first-generation tyrosine kinase inhibitors (TKIs), with exon 20 patients presenting response rates of only 4-8% and a median progression free survival (mPFS) of 2 months based on retrospective studies. Similarly, patients with atypical EGFR exon 18 mutations have less clinical benefit than patients with classical EGFR mutations when treated with common EGFR- TKIs. The population impacted by atypical EGFR NSCLC mutations is sizable: approximately 5,000 patients per year in the US, 41,600 patients per year worldwide, and an even greater number of patients outside of NSCLC. We recently reported the results of a detailed structural and functional analysis that led to the identification of the TKI, poziotinib, as a potent and clinically active inhibitor of EGFR and HER2 exon 20 mutant tumors. Based on this data we have conducted an investigator-initiated phase II trial testing poziotinib in EGFR and HER2 exon 20 mutant NSCLC patients, demonstrating that it is highly active drug for with confirmed objective responses observed in 43% of patients. This clearly represents an advance for these patients, however, the drug has significant limitations. Responses to poziotinib treatment were of limited duration, with a mPFS of 5.5 months, and even less activity was observed in patients with mutations in the ?far loop? of exon 20. Whereas patients with classical EGFR mutations have a mPFS of 18.9 months to osimertinib. Furthermore, many patients receiving poziotinib experienced >grade 3 adverse events, diarrhea and rash, related to the activity of this drug against wild-type EGFR, resulting in dose reduction in >60% of patients. To address these limitations, we propose an integrative analysis to understand the structural features of EGFR exon 18 and 20 mutations, so that more potent inhibitors can be developed and that specificity for mutant to WT receptor can be enhanced. To achieve this, we propose the following aims: Aim 1) we will study the structural features of EGFR exon 20 mutations that drive differential sensitivity to TKIs to guide the development of novel compounds with increased potency. Aim 2) we will analyze the structural features of atypical EGFR exon 18 mutations to use this information to rationally design new compounds with enhance efficacy. Aim 3) we will develop novel TKIs with improved EGFR mutant vs wild- type specificity to augment drug tolerability and clinical benefit of EGFR exon 18 and exon 20 mutant patients. We have assembled a unique, multi-disciplinary team of physicians, laboratory scientist, structural biologist, and medicinal chemists with unparalleled pre-clinical and clinical resources for who will work cooperatively to gain insights into the structural features of EGFR exon 18 and 20 mutant cancers and to translate this into preclinical and potentially clinical testing. Furthermore, insights gained from this study can help to accelerate efforts worldwide aiming to target EGFR exon 18 and 20 tumors as well as other EGFR mutant caners.